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Flow cytometric analysis of MGL1 and MGL2 expression by the use of specific monoclonal antibodies
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Flow cytometric analysis of MGL1 and MGL2 expression by the use of specific monoclonal antibodies

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Sugar binding proteins
Protocol Name

Flow cytometric analysis of MGL1 and MGL2 expression by the use of specific monoclonal antibodies

Authors
 Murakami, Ryuichi
Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, The University of Tokyo

Denda-Nagai, Kaori
Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, The University of Tokyo

Irimura, Tatsuro *
Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, The University of Tokyo
*To whom correspondence should be addressed.
KeyWords
Reagents
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Preparation of monoclonal antibodies specific for MGL1 or MGL2 is described elsewhere (Denda-Nagai, K et al. 2010; Kimura, T et al 1995).

Methods
1.

Cell preparation from peripheral lymph nodes

1) 

 Axillary, brachial, and inguinal lymph nodes (LNs) are obtained from naïve mice.

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2) 

 LNs are minced and digested for 20 min at 37°C with 1 mg/mL collagenase from Clostridium histolyticum (Sigma-Aldrich, St. Louis, MO) in cRPMI (RPMI 1640 medium supplemented with 10% FCS and 10 mM HEPES).

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3) 

 The digested LNs are re-suspended by pipetting.

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4) 

 Phosphate buffered saline (PBS) is added to LN cell suspensions.

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5) 

 LN cell suspensions are transferred to new tube through iron mesh and centrifuged.

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6) 

 LN cells are treated with 10 mM EDTA for 5 min to inactivate collagenase and to disrupt T cell-DC complexes.

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7) 

 PBS is added to LN cell suspensions.

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8) 

 LN cell suspensions are transferred to a new tube through nylon mesh and centrifuged.

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9) 

 LN cells are washed twice with PBS.

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10) 

 LN cells are suspended in PBS containing 0.1% BSA and 0.1% sodium azide (FCM buffer).

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2.

Staining of isolated LN cells and flow cytometric analysis

1) 

 Cells are incubated with anti-mouse CD16/CD32 (1/100 dilution of ammonium sulfate precipitated hybridoma culture supernatants of 2.4G2 hybridoma purchased from ATCC) to reduce nonspecific binding 5 min before the addition of the first antibodies.

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2) 

 Cells are incubated with biotin-conjugated anti-MGL1 mAb LOM8.7 (bio-LOM8.7: 10 μg/mL), allophycocyanin-conjugated anti-MGL2 mAb URA-1 (APC-URA-1: 10 μg/mL), PE-Cy5-conjugated anti-CD3 (0.4 μg/mL) and PE-Cy5-conjugated anti-CD19 (eBioscience: 0.1 μg/mL) for 30 min on ice.

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3) 

 Cells are washed with FCM buffer and centrifuged.

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4) 

 Supernatants are removed and tubes are tapped until cells are separated.

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5) 

 Cells are incubated with 0.2 μg/mL PE-Cy7-labeled streptavidin (SAV) (BioLegend, San Diego, CA) for 30 min on ice to visualize biotin-conjugated mAb LOM8.7.

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6) 

 Cells are washed with FCM buffer and centrifuged.

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7) 

 Supernatants are removed and tubes are tapped until cells are separated.

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8) 

 Cells are re-suspended in FCM buffer containing 1 μg/mL 7-amino-actinomycin D (eBioscience, Inc., San Diego, CA) to exclude dead cells.

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9) 

 Samples are analyzed on FACSAria cell sorter (BD, Franklin Lakes, NJ).

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10) 

 Data are analyzed using FlowJo software (Tree Star, Ashland, OR).

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